We now recognize three distinct motifs of DNA-binding zinc proteins: (i) fingers, (ii) clusters, and (iii) twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses proteins have not been available to serve as standards reference for the binding sites these families proteins. Those domains fungal transcription factor GAL4 rat glucocorticoid receptor are first determined. Both contain two sites, in both, cysteine residues sole ligands. In GAL4, atoms...

The DNA-binding domain of the transcription factor GAL4, consisting 62 N-terminal residues and denoted GAL4(62*), contains a Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys motif, which has been shown previously to bind two Zn(II) or Cd(II) ions. Binding is essential for recognition by GAL4 specific palindromic DNA sequence it binds upstream genes galactose-metabolizing enzymes, UASG sequence. On basis 113Cd NMR chemical shifts bound 113Cd(II) ions, we propose binuclear cluster model...

Abstract Reversible, pH-dependent changes in the secondary, tertiary, and quaternary structure of Escherichia coli alkaline phosphatase have been studied by optical rotatory dispersion, circular dichroism, sedimentation velocity equilibrium centrifugation, with use native zinc enzyme, apoenzyme, an enzyme which has replaced cobalt. Ultracentrifugation studies show that molecule remains a dimer between pH 8 4. The weight average molecular weight, Mw, as determined Yphantis method over this...

Almost all known intracellular fusion reactions are driven by formation of trans -SNARE complexes through pairing vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number SNARE required for is unknown, and there controversy about whether additional proteins to explain fast which can occur in cells. Here we show that single vesicles containing synaptic/exocytic VAMP/synaptobrevin fuse rapidly planar, supported bilayers syntaxin-SNAP25. Fusion rates...

Myo4p is a nonessential type V myosin required for the bud tip localization of ASH1 and IST2 mRNA. These mRNAs associate with via She2p She3p proteins. an adaptor protein that links to its cargo. binds mRNA, while both Myo4p. Here we show She3p, but not She2p, are inheritance cortical ER in budding yeast Saccharomyces cerevisiae. Consistent this observation, find independent mRNA transport. Cortical dynamic network forms cytoplasmic tubular connections nuclear envelope. tubules failed grow...

Gene 32 protein (g32P) isolated from bacteriophage T4-infected Escherichia coli and an overproduction vector derived the plasmid pKC30 contains 1 mol of tightly incorporated Zn(II) per protein. A linear incorporation three molar equivalents p-hydroxymercuriphenylsulfonate (PMPS) results in a release 1.1 Reversal formation g32P-PMPS complex with thiol presence EDTA zinc-free apo-g32P. Cd(II) Co(II) can be exchanged intrinsic ion. The shows charge-transfer band at approximately 250 nm. set...

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta a systematic screen for yeast mutants with altered ER morphology. cells is predominantly rather than reticular, yet the net surface area of not significantly changed. Rtn1-green fluorescent protein (GFP) associates at cell cortex tubules that connect cortical to nuclear envelope, but envelope itself. Rtn1p overexpression also results an Rtn proteins are found on...

Abstract A system for detecting the effects of metal ions, repressors, and mutations on biosynthesis in vivo alkaline phosphatase Escherichia coli is described. If E. constitutive are grown a medium containing 65Zn subject to osmotic shock, about 50% zinc taken up by cells released into medium. Chromatography this shows that most associated with phosphatase. The ratio specific radioactivity enzyme 2 3 g atoms incorporated per mole under these conditions. This biosynthetically not...

Monocular lid closure (MC) causes a profound shift in the ocular dominance (OD) of neurons primary visual cortex (V1). Anatomical studies both cat and mouse V1 suggest that large-scale structural rearrangements eye-specific thalamocortical (TC) axons response to MC occur much more slowly than OD. Consequently, there has been considerable debate as whether plasticity TC synapses, which transmit competing information from each eye V1, contributes early functional consequences or is simply...

Significance Synaptotagmin-1 is the calcium sensor for synchronous neurotransmitter release. It couples influx to soluble N -ethylmaleimide–sensitive factor activating protein receptor (SNARE)-catalyzed fusion, but how this coupling happens unknown. Here, using electron microscopy, we report that cytosolic domain of synaptotagmin can assemble into ring-like oligomers under calcium-free conditions, and these rings disassemble rapidly upon binding. This process suggests a novel speculative...

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes regulated and function. To determine mechanisms, we characterized the folding energy kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found all assemble by same step-wise zippering mechanism: slow N-terminal...

Mutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed regulate neurotransmitter release. However, mechanism action unclear. Here, we use reconstituted single vesicle bulk fusion assays, combined live cell imaging exocytotic events PC12 cells biophysical analysis, delineate physiological role PRRT2. We report that selectively blocks trans SNARE...

Significance Synaptic vesicle fusion at synapses is the primary mechanism by which neurons communicate. A highly conserved membrane machine known as SNARE complex mediates this process. In addition, neuronal SNARE-binding regulatory proteins have evolved to control kinetics and speed of assembly synapses. One such protein, Complexin, has been found inhibit synaptic in absence an action potential activate SNARE-mediated release during stimulation. Here we examine molecular models for how...

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate fusion by pulling biological membranes together via a zippering mechanism. Recent biophysical studies have shown that t- and v-SNAREs can assemble in multiple stages from the N-termini toward C-termini. Here we show functionally, membrane requires sequential, two-step folding pathway assign specific distinct functions for each step. First, N-terminal domain (NTD) of v-SNARE docks to t-SNARE, which...

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which potentially regulate neurotransmitter release. Here we report analogous assemble from domains other Syt isoforms (Syt2, Syt7, Syt9) as well related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general conserved structural aspect many proteins, including Synaptotagmins....

Significance Release of neurotransmitters relies on submillisecond coupling synaptic vesicle fusion to the triggering signal: AP-evoked presynaptic Ca 2+ influx. The key player that controls exocytosis is sensor synaptotagmin 1 (Syt1). While activation Syt1 has been extensively characterized, how reversibly clamps vesicular remains enigmatic. Here, using a targeted mutation combined with fluorescence imaging and electrophysiology, we show structural feature self-oligomerize provides...

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion (clamping) and trigger following Ca2+-influx. The principal components involved in these processes are the vesicular machinery (SNARE proteins) regulatory proteins, Synaptotagmin-1 Complexin. Here, we use a reconstituted single-vesicle assay under physiologically-relevant conditions delineate novel mechanism by which Complexin act synergistically establish...

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate maintain a pool of docked vesicles fuse rapidly synchronously upon Ca 2+ influx (Ramakrishnan et al., 2020). Here, using same in vitro single-vesicle fusion assay, determine molecular details Complexin-mediated its role -activation. We find delay kinetics, likely imparted by Synaptotagmin-1, is needed for block fusion. Systematic truncation/mutational analyses reveal...

The synaptic vesicle protein Synaptophysin (Syp) has long been known to form a complex with the Vesicle associated soluble N-ethylmaleimide sensitive fusion attachment receptor (v-SNARE) membrane (VAMP), but more specific molecular function or mechanism of action in exocytosis lacking because gene knockouts have minimal effects. Utilizing fully defined reconstitution and single-molecule measurements, we now report that Syp functions as chaperone determines number SNAREpins assembling between...

31P NMR spectra of phosphate and phosphonate complexes Escherichia coli alkaline phosphatase have been obtained by Fourier transform methods. One equivalent P1i, bound to Zn(II) phosphatase, pH 8, gives rise a single resonance 2 ppm downfield from that for Pi, assignable the noncovalent complex, E-P. Inorganic in excess 1 eq per enzyme dimer at position expected free Pi. At 5.1, second appears 8.5 is covalent The large shift suggests phosphoryl group highly strained with an O-P-O bond angle...

Abstract Optical rotatory dispersion and circular dichroism spectra of the Pseudomonas blue protein laccase B from Polyporus versicolor are presented 190 to 700 mµ. Circular both proteins show six ellipticity bands above 300 mµ associated with copper chromophore, indicating more transitions be present in these complexes than three parity-forbidden d-d Cu(II) its common symmetries. A Gaussian analysis visible is presented. The same set generates absorption by adjustment signs heights bands,...